Development of a gene synthesis platform for the efficient large scale production of small genes encoding animal toxins

BMC Biotechnology

Methodology Article

Carlos M. G. A. Fontes¹  Ana Filipa Sequeira¹  Catarina I. P. D. Guerreiro²  Joana L. A. Brás²  Renaud Vincentelli³ 

[1] Centro Interdisciplinar de Investigação em Sanidade Animal (CIISA) - Faculdade de Medicina Veterinária, Universidade de Lisboa, Avenida da Universidade Técnica, 1300-477, Lisboa, Portugal;NZYTech Genes & Enzymes, Campus do Lumiar, Estrada do Paço do Lumiar, Edifício E, r/c, 1649-038, Lisboa, Portugal;NZYTech Genes & Enzymes, Campus do Lumiar, Estrada do Paço do Lumiar, Edifício E, r/c, 1649-038, Lisboa, Portugal;Unité Mixte de Recherche (UMR) 7257, Centre National de la Recherche Scientifique (CNRS) – Aix-Marseille Université, Architecture et Fonction des Macromolécules Biologiques (AFMB), Campus de Luminy, 163 Avenue de Luminy, 13288, Marseille, CEDEX 09, France;

关键词: Gene synthesis;    Assembly PCR;    Gene design;    Venom peptides;   

DOI  :  10.1186/s12896-016-0316-3

 received in 2016-04-16, accepted in 2016-11-23,  发布年份 2016

来源: Springer

【 摘 要 】

BackgroundGene synthesis is becoming an important tool in many fields of recombinant DNA technology, including recombinant protein production. De novo gene synthesis is quickly replacing the classical cloning and mutagenesis procedures and allows generating nucleic acids for which no template is available. In addition, when coupled with efficient gene design algorithms that optimize codon usage, it leads to high levels of recombinant protein expression.ResultsHere, we describe the development of an optimized gene synthesis platform that was applied to the large scale production of small genes encoding venom peptides. This improved gene synthesis method uses a PCR-based protocol to assemble synthetic DNA from pools of overlapping oligonucleotides and was developed to synthesise multiples genes simultaneously. This technology incorporates an accurate, automated and cost effective ligation independent cloning step to directly integrate the synthetic genes into an effective Escherichia coli expression vector. The robustness of this technology to generate large libraries of dozens to thousands of synthetic nucleic acids was demonstrated through the parallel and simultaneous synthesis of 96 genes encoding animal toxins.ConclusionsAn automated platform was developed for the large-scale synthesis of small genes encoding eukaryotic toxins. Large scale recombinant expression of synthetic genes encoding eukaryotic toxins will allow exploring the extraordinary potency and pharmacological diversity of animal venoms, an increasingly valuable but unexplored source of lead molecules for drug discovery.

【 授权许可】

CC BY   
© The Author(s). 2016